In recent years, it has become increasingly desirable and important in biological research to freeze cell cultures so that they may be stored in the frozen state until immediately prior to use because viability of the cells can thus be maintained for extended periods of time. This freezing and storage process contemplates that cell cultures at room temperatures of about +20.degree. C. be reduced in temperature to temperatures of at least -30.degree. C. or lower.
However, the freezing of such cultures is not without certain shortcomings. Cell cultures typically are suspended in a water based medium for growth and storage. As known in the art, cryogenic preservation and storage of cell cultures requires the inclusion of dimethyl sulfoxide or glycerol. When freezing this water based suspension to preserve the viability of the cells, damage can occur to the cells if there is a formation of ice crystals which can puncture or otherwise physically damage the cell walls. This potential problem can be minimized by rapidly freezing the culture. This rapid freezing promotes the formation of small ice crystals which generally do not cause as much damage to the cells as larger ice crystals, typically formed during slower freezing.
On the other hand, too abrupt a change in the physical state of the cells, as by too rapid a freezing of the culture, can destroy the cells to be stored or otherwise have an adverse effect on subsequent cell activity. Hence, it is generally recognized by those skilled in the art that optimum freezing of cell cultures, particularly eucaryotic cell cultures, from room temperature to at least -30.degree. C. or lower is achieved if the temperature of the cell culture decreases at a theoretical rate of approximately 1.degree. C. per minute. Freezing at this theoretically preferred -1.degree. C./min. rate is generally recognized as the optimum temperature reduction for cryopreservation of live cells, especially eucaryotic cells. This optimum temperature reduction is most critical in the range of about -4.degree. C., when cultures begin crystal formations, to about -25.degree. C., where crystal formation is complete.
The equipment presently available to research biologists and others for freezing cell cultures is extremely expensive or unreliable in controlling the freezing of the cultures. One type equipment employs liquid nitrogen as the freezing medium and generally costs in excess of $10,000. One such device is manufactured by Planer Products Ltd. of Great Britain and identified as the Planer Mini-Freezer R202/200R. A much lower priced alternative device is manufactured by Union Carbide Corporation and is identified as the "BF-5 Biological Freezer". This device, however, has the distinct disadvantage of requiring the research scientist to correctly calibrate the position of the vial holder within the freezing compartment, depending on the number of individual cell cultures to be frozen. Such calibration often results in inconsistent freezing of the cultures.
In view of the extremely high cost of the only reliable presently available freezing equipment, individual research scientists have employed their own homemade controlling devices for use in their conventional laboratory freezers, which are normally operated at -70.degree. C. to -90.degree. C. Such homemade devices have included cotton wrapping around the cell culture vials which are then placed into a styrofoam box, or jars containing alcohol. However, these homemade devices have not been satisfactory since they produce inconsistent controlled freezing and generally fail to approach the desired theoretical optimum temperature reduction. Furthermore, they very often fail to maintain the proper orientation of the stored cell culture vials during the freezing process.
Accordingly, it is the principal object of this invention to provide a simple, inexpensive, but very reliable device for controlling the freezing of cell cultures in conventional laboratory freezers, especially eucaryotic cell cultures.
A further object of this invention is to provide a device for freezing cell cultures which approaches the optimum -1.degree. C./min. freezing rate which promotes the best preservation of viability of the live cells.
Another object resides in the novel supporting mechanism of the freeze controlling device by which the vials containing the cell cultures are easily and safely supported and stored within the device.
A still further important object of the present invention is to provide uniform temperature reduction of the cultures without variation or calibration of the device, regardless of the number of different cultures being frozen at the same time.
The final important object of this invention is to provide a method by which controlled freezing of live cell cultures can be easily and safely carried out in standard laboratory freezers operated at -70.degree. C., or below.